Use of an acidic aqueous solution of a bioadhesive polyphenolic protein as an adhesive or coating

ABSTRACT

The present invention pertains to the use of an acidic aqueous solution of a bioadhe-sive polyphenolic protein, derived from a byssus-forming mussel, for attaching two surfaces to each other or coating a surface, which acidic solution has a pH of 4 or less and in which the concentration of the bioadhesive protein is between 10-250 mg/ml. The acidic aqueous solution of the polyphenolic protein is added directly as a sole component to the surfaces to be attached to each other or the surface to be coated.

The present invention pertains to the direct use of an acidic aqueoussolution of a bioadhesive protein for attaching two surfaces to eachother or coating a surface.

BACKGROUND OF THE INVENTION

Attachment of different structures is crucial in a wide variety ofprocesses. However, this is frequently associated with problems ofdifferent nature depending on what structures are to be attached.

Areas that are particularly troublesome are adhesion in the medicalfield, and attachment of components of very small size, such as in themicro- and nano-techniques. In the medical field, examples of whenadhesives have to be used to adhere biological material include repairof lacerated or otherwise damaged organs, especially broken bones anddetached retinas and corneas. Dental procedures also often requireadhesion of parts to each other, such as during repair of caries,permanent sealants and periodontal surgery. It is very important inbiomedical applications of an adhesive and coating composition to usebioacceptable and biodegradable components, which furthermore should notper se or due to contamination induce any inflammation or toxicreactions. In addition, the adhesive has to be able to attach structuresto each other in a wet environment.

In the electronic industry, a particular problem today is that thecomponents that are to be attached to each other often are of very smallsize, and the amount of adhesive that is possible to use is very small.Adhesives that provide high adhesive strength even with minor amounts ofadhesive are therefore required. Also for non-medical uses, an adhesivethat is non-irritating, non-allergenic, non-toxic and environmentallyfriendly is preferred. However many of the commonly used adhesivesinduce toxic reactions in the user, for example due to their contents oforganic solvent.

Polyphenolic proteins, preferentially isolated from mussels, are knownto act as adhesives. Examples of such proteins can be found in e.g. U.S.Pat. No. 4,585,585. Their wide use as adhesives has been hampered byproblems related to the purification and characterisation of theadhesive proteins in sufficient amounts. Also, mostly when using thepolyphenolic proteins as adhesives the pH has had to be raised toneutral or slightly basic in order to facilitate oxidation and curing ofthe protein. However, this curing is slow and results in poor adhesivestrength and therefore oxidisers, fillers and cross-linking agents arecommonly added to decrease the curing time and obtain a strongeradhesive. In addition, in an earlier study (EP-A-244 688) the adhesivestrength using, as a sole component without raising the pH, an acidicsolution of MAP (5 mg/ml in 5% acetic acid) was demonstrated to be poor,compared to when a filler protein was added to the composition beforeadhesion (2.5 mg/ml each of MAP and casein).

Mussel adhesive protein (MAP) is formed in a gland in the foot ofbyssus-forming mussels, such as the common blue mussel (Mytilus edulis).The molecular weight of MAP from Mytilis edulis is about 130.000 Daltonand it has been disclosed to consist of 75-80 closely related repeatedpeptide sequences. The protein is further characterised by its manyepidermal growth factor like repeats. It has an unusual high proportionof hydroxy-containing amino acids such as hydroxyproline, serine,threonine, tyrosin, and the uncommon amino acid3,4-dihydroxy-L-phenylalanine (Dopa) as well as lysine. It may beisolated either from natural sources or produced biotechnologically.U.S. Pat. No. 5,015,677 as well as U.S. Pat. No. 4,585,585 disclose thatMAP has very strong adhesive properties after oxidation andpolymerisation, e.g. by the activity of the enzyme tyrosinase, or aftertreatment with bifunctional reagents.

MAP is previously known to be useful as an adhesive composition e.g. forophthalmic purposes. Robin et al., Refractive and Corneal Surgery, vol.5, p. 302-306, and Robin et al., Arch. Ophthalmol., vol. 106, p.973-977, both disclose MAP-based adhesives comprising an enzymepolymiser. U.S. Pat. No. 5,015,677 also describes a MAP-based adhesivecontaining a cross-linking agent and optionally a filler substance and asurfactant. Preferred cross-linking agents according to U.S. Pat. No.5,015,677 are enzymatic oxidising agents, such as catechol oxidase andtyrosinase, but sometimes also chemical cross-linking agents, such asglutaraldehyde and formaldehyde can be used. Examples of fillers areproteins, such as casein, collagen and albumin, and polymers comprisingcarbohydrate moieties, such as chitosan and hyaluronan. U.S. Pat. No.5,030,230 also relates to a bioadhesive comprising MAP, mushroomtyrosinase (cross-linker), SDS (sodium dodecyl sulfate, a surfactant)and collagen (filler). The bioadhesive is used to adhere a corneaprosthesis to the eye wall.

EP-A-343 424 describes the use of a mussel adhesive protein to adhere atissue, cell or another nucleic acid containing sample to a substrateduring nucleic acid hybridisation conditions, wherein the musseladhesive protein, despite the harsh conditions encountered during thehybridisation, provided adherence. U.S. Pat. No. 5,817,470 describes theuse of mussel adhesive protein to immobilise a ligand to a solid supportfor enzyme-linked immunoassay. Mussel adhesive protein has also beenused in cosmetic compositions to enhance adherence to nails and skin (WO88/05654).

A major problem associated with known MAP-based bioadhesivecompositions, despite the superior properties of MAP per se, is thatsome constituents, in particular commonly used cross-linking agents, canharm and/or irritate living tissue and cause toxic and immunologicalreactions. Chemical crosslinking agents, such as glutaraldehyde andformaldehyde, are generally toxic to humans and animals, and it ishighly inappropriate to add such agents to a sensitive tissue, such asthe eye. Enzymes, such as catechol oxidase and tyrosinase, are proteins,and proteins are generally recognised as potential allergens, especiallyin case they originate from a species other than the patient. Because oftheir oxidising and hydrolysing abilities, they can also harm sensitivetissue.

Therefore, there is still a need for adhesive compositions, both formedical and other applications, that provide strong adhesion with smallamounts of adhesive, that are simple to use and that do not cause toxicand allergic reactions.

SUMMARY OF THE INVENTION

The present invention pertains to the use of an acidic aqueous solutionof a bioadhesive polyphenolic protein, derived from a byssus-formingmussel, for attaching two surfaces to each other or coating a surface,which acidic solution has a pH of 4 or less and in which theconcentration of the bioadhesive protein is between 10-250 mg/ml. Theuse of this acidic solution of the bioadhesive protein as a solecomponent avoids the addition of additional components to effectadhesion and therefore the process of adhesion is simplified and therisk of causing allergy and/or irritation due to the additionalcomponents added is decreased. The composition is therefore well-suitedfor medical application. Also, the adhesive strength obtained is high,even with small amounts of adhesive, and the composition is thereforealso preferably used when only small amounts of adhesive can be appliedto surfaces to be joined or coated. The composition of the presentinvention is also suitable for use in wet environments.

DEFINITIONS

As disclosed herein, the terms “polyphenolic protein”, “mussel adhesiveprotein” or “MAP” relates to a bioadhesive protein derived frombyssus-forming mussels or which is recombinantly produced. Examples ofsuch mussels are mussels of the genera Mytilus, Geukensia, Aulacomya,Phragmatopoma, Dreissenia and Brachiodontes. Suitable proteins have beendisclosed in a plurality of publications, e.g. U.S. Pat. No. 5,015,677,U.S. Pat. No. 5,242,808, U.S. Pat. No. 4,585,585, U.S. Pat. No.5,202,236, U.S. Pat. No. 5,149,657, U.S. Pat. No. 5,410,023, WO97/34016, and U.S. Pat. No. 5,574,134, Vreeland et al., J. Physiol., 34:1-8, and Yu et al., Macromolecules, 31: 4739-4745. They comprise about30-300 amino acid residues and essentially consist of tandemly linkedpeptide units comprising 3-15 amino acid residues, optionally separatedby a junction sequence of 0-10 amino acids. A characteristic feature ofsuch proteins is a comparatively high amount of positively chargedlysine residues, and in particular the unusual amino acid DOPA(L-3,4-dihydroxyphenylalanine). A polyphenolic protein suitable for usein the present invention has an amino acid sequence in which at least 3%and preferably 6-30% of the amino acid residues are DOPA. A few examplesof typical peptide units are given below. However, it is important tonote that the amino acid sequences of these proteins are variable andthat the scope of the present invention is not limited to theexemplified subsequences below, as the skilled person realises thatbioadhesive polyphenolic proteins from different sources, includingrecombinantly produced, can be regarded as equivalent:

-   a) Val-Gly-Gly-DOPA-Gly-DOPA-Gly-Ala-Lys-   b) Ala-Lys-Pro-Ser-Tyr-diHyp-Hyp-Thr-DOPA-Lys-   c) Thr-Gly-DOPA-Gly-Pro-Gly-DOPA-Lys-   d) Ala-Gly-DOPA-Gly-Gly-Leu-Lys-   e) Gly-Pro-DOPA-Val-Pro-Asp-Gly-Pro-Tyr-Asp-Lys-   f) Gly-Lys-Pro-Ser-Pro-DOPA-Asp-Pro-Gly-DOPA-Lys-   g) Gly-DOPA-Lys-   h) Thr-Gly-DOPA-Ser-Ala-Gly-DOPA-Lys-   i) Gln-Thr-Gly-DOPA-Val-Pro-Gly-DOPA-Lys-   j) Gln-Thr-Gly-DOPA-Asp-Pro-Gly-Tyr-Lys-   k) Gln-Thr-Gly-DOPA-Leu-Pro-Gly-DOPA-Lys

The term “surface” is to be interpreted broadly and may comprisevirtually any surface. The choice of surface is not critical to thepresent invention. Examples of surfaces for which the invention arespecially suitable for include non-biological surfaces such as glass,plastic, ceramic and metallic surfaces etc., and biological surfaces,comprising wood and different tissues such as skin, bone, teeth, theeye, cartilage, etc.

By acidic aqueous solution is meant an aqueous solution comprising anorganic or inorganic acid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention describes the use of a polyphenolic bioadhesivecomposition to attach two surfaces to each other or coating a surface.The compositions provided in the invention can in principle be used toattach any surfaces to each other or to coat any surface. However, thecompositions according to the present invention are particularly usefulwhen adhesive or coating compositions are needed that are non-toxic,non-irritating or non-allergenic, since the only mandatory component isthe bioadhesive protein in itself and this has a low risk of causingsuch reaction. The use of a bioadhesive composition described in thepresent invention allows very small amounts of adhesive to be used whilestill achieving a strong adhesion. Therefore the use of the compositionof the present invention is particularly useful when only small amountsof adhesive can be used. Further advantages with the use of thecomposition provided in the present invention are their watersolubility, the avoidance of organic solvents commonly used in adhesiveor coating compositions, and that they are biologically produced andharmless to the environment.

The only mandatory component of the present invention is thepolyphenolic protein itself provided in an acidic solution, for examplethe same acidic solution that is used for storage of the protein.Previously when polyphenolic proteins have been used, it has beenthought to be necessary to add additional components, such as fillersand oxidising agents and/or raise the pH to neutral or slightly basic,in order to achieve strong enough adhesive strength. The presentinventor has shown that a very strong adhesion, comparable to theadhesive strength provided using the commonly used MAP compositions, canbe provided employing a concentrated acidic MAP-solution directly.Therefore, since no additional components have to be added to theMAP-solution before its use, the process of adhesion is simplified overearlier uses of bioadhesive proteins. Also due to the simple compositionof the adhesive, the risks of irritation and/or allergy that have beencommon with earlier uses of bioadhesive polyphenolic proteins isavoided.

According to the present invention the acidic solution of theMAP-protein is applied, as a sole component, to at least one of thesurfaces, which are to be attached to each other, before the surfacesare joined, or added to the surface to be coated. The composition of theinvention was demonstrated to cure both in dry and wet environments. Ascan be seen in the appended Examples the curing time can be as short as1 min.

The concentration of the MAP-solution of the present invention is 10-250mg/ml. Preferably the concentration of the MAP-solution is 10-150 mg/ml.More preferably the MAP-concentration is 30-100 mg/ml and mostpreferably 40-80 mg/mL. It is important that the concentration of theMAP-solution is at least 10 mg/ml, since earlier experiments have showna poor adhesive strength using a 5 mg/ml MAP-solution in 5% acetic acid(EP-A-244 688), if no additional components were added to effect curing.

The MAP protein of the present invention is provided in an acidicaqueous solution with a pH of 4 or less. However, a pH of 3 or less wasalso unexpectedly found to result i high adhesive strengths. Even moresurprisingly at pH of 2.5 or less was found to result in high adhesivestrengths. Acids suitable for the present invention include bothinorganic acids, such as hydrochloric acid and phosphoric acid, andorganic acids, such as citric acid, ascorbic acid, and acetic acid. Onepreferred object of the present invention is to provide an adhesive orcoating composition for medical applications, e.g. for attachingbiological and non-biological components to biological structures, anobject for which the MAP-protein in itself is well suited, since it isnon-toxic and biodegradable. However, the components commonly added toMAP-compositions in order to obtain cross-linking and oxidation(chemical and/or enzymatic crosslinkers and oxidising agents) of thecomposition can lead to irritation and allergic reactions and thoseMAP-compositions are therefore not optimal for medical applications. Dueto the lack of such components in the present invention, thecompositions of the present invention are particularly suitable forattachment of biological surfaces to each other or to other,non-biological, materials. Also, since only small amounts of theadhesive composition of the present invention is required, while stillproviding high adhesive strengths, the composition of the presentinvention is particularity suitable for medical applications where oftenonly small amounts of adhesives can be applied to surfaces to be adheredto each other or surfaces to be coated. For the above reasons, the useof the composition of the present invention is particularily suitablefor adhesion of corneas, tendons, tissues during surgical operation etc.For the above reasons, the compositions of the present invention arealso particularly useful for coating of materials used in medicalapplications or biological tissues.

Due to the very high adhesive strength provided with very small amountsof the compositions of the present invention, one preferred field ofapplication for which the compositions are particularly suitable is forattachment of non-biological surfaces such as glass, plastic, ceramicand metallic surfaces. This is particularly useful within the electronicmicro- and nano-techniques, optics, etc. for adhesion or coating ofcomponents in, for example, biosensors, microchips, solar cells, mobilephones, etc., since for these applications only minute amounts ofadhesive can be used. The compositions of the present invention are alsosuitable for coating of non-biological surfaces.

The adhesive compositions of the present invention are also useful forattachment of cells, enzymes, antibodies and other biological specimento surfaces.

EXAMPLE 1

In order to determine the adhesive strength using the compositions ofthe present invention, the adhesive strength between non-biologicalmaterial (glass plates, 75×25×2 mm) and biological tissue (muscle fromcattle and pig) was determined. The aqueous, acidic MAP-solution withvarying concentrations (see Table 1) in 0.01 M citric acid (pH ca 2.3)was applied to one of the surfaces that were to be attached to eachother before joining the two surfaces and fixing them with a clip. Thesamples were thereafter allowed to cure for different time periods andunder different conditions before the adhesive strength was determinedusing a digital spring balance (Milo) by attaching either the glassplate or the biological tissue to the balance and thereafter stretchinguntil the glass plate and biological tissue were detached from eachother. The adherence surfaces were in most cases 0.2-0.4 cm², with avariation from 0.1 to 0.8 cm². As can be seen in the results in Table 1the adhesive strength is not weakened when the samples are allowed tocure under wet conditions, even though no cross-linking agent isemployed. TABLE 1 Adhesive strength achieved between biological andnon-biological surfaces using the MAP-composition of the presentinvention. MAP MAP Concentration Amount Acid Sample (mg/ml) (μg)(concentration) Curing conditions Adhesive strength (g) 1 23 69 Citricacid (0.01 M) 24 h in water at 4° C. 40 2 25 75 Citric acid (0.01 M) 24h in water at 4° C. 45 3 20 60 Citric acid (0.01 M) 1 h in water at 35°C. 60 4 20 60 Citric acid (0.01 M) 1 h in water at 35° C. 40 5 24 72Citric acid (0.01 M) 1 min under dry condi- 40 tions

EXAMPLE 2

In order to determine the adhesive strength using the compositions ofthe present invention, the adhesive strength between biological tissue(muscle from cattle and pig) was determined. The acidic MAP-solution(see Table 2) in 0.01 M citric acid (pH ca 2.3) was applied to one ofthe surfaces that were to be attached to each other before joining thetwo surfaces and fixing them with a clip. The samples were thereafterallowed to cure under water at 35° C. before the adhesive strength wasdetermined using a digital spring balance (Milo) by attaching one of thetwo parts of biological tissue to the balance and thereafter stretchinguntil the biological tissues were detached from each other. Theadherence surfaces were in most cases 0.2-0.4 cm², with a variation from0.1 to 0.8 cm². TABLE 2 Adhesive strength achieved between biologicalsurfaces using the MAP-composition of the present invention. MAP MAPConcentration Amount Acid Sample (mg/ml) (μg) (concentration) Curingconditions Adhesive strength (g) 1 20 50 Citric acid (0.01 M) 1 hour inwater at 35° C. 100 2 18 45 Citric acid (0.01 M) 1 hour in water at 35°C. 120

EXAMPLE 3

To determine the adhesive strength achieved between two non-biologicalsurfaces, two glass plates (ca 75×25×1.5 mm) were attached to each otherby placing a droplet of acidic MAP-solution on one of the glass plates,placing the other glass plate on top of the first and fixing the twoglass plates to each other using a clip. The concentrations and amountsof the MAP-solutions employed are specified in Table 3 below, as is theacid, and its concentration, that is used for each specific experiment.The pH-values for the different acids employed were as follows: 0.05 Mcitric acid: pH ca 1.8; 0.01 M citric acid: pH ca 2.3; 0.2 M aceticacid: pH ca 2.3; 0.014 M ascorbic acid: pH 2.9; 0.05 M HCl: pH ca 1.0;and 0.05 M H₃PO₄: pH ca 1.4. The samples were left for 24 hours at roomtemperature before determining adhesive strength. The adhesive strengthwas determined by measurement of shear strength (see Table 3) employingconventional techniques. The adhesive area varied between 0.3-1.0 cm².As a comparison the adhesive strength employing standard epoxy glue wasdetermined. Use of 10 mg of this to the glass plates in a similarfashion as described above resulted in an adhesive strength of 380 N.TABLE 3 Adhesive strength achieved between non-biological surfaces usingthe MAP-composition of the present invention. MAP MAP Acid Adhesivestrength Sample Concentration (mg/ml) Amount (μg) (concentration) (N) 124 48 Citric acid (0.01 M) 226 2 42 42 Citric acid (0.05 M) 290 3 42 84Citric acid (0.05 M) 430 4 39 117 Citric acid (0.05 M) 401 5 39 117Citric acid (0.05 M) 437 6 42 42 Citric acid (0.05 M) 350 7 27 54 Aceticacid (0.2 M) >240 8 28 56 Ascorbic acid (0.014 M) 353 9 28 56 Ascorbicacid (0.014 M) 328 10 23 46 HCI (0.05 M) >270 11 25 50 H₃PO₄ (0.05 M)237

1. A composition for attaching two surfaces to each other or for coatinga surface, consisting of: an acidic aqueous solution of a bioadhesivepolyphenolic protein derived from a byssus-forming mussel which proteincomprises 30-300 amino acids and consists essentially of tandemly linkedpeptide repeats comprising 3-15 amino acid residues, wherein at least 3%of the amino acid residues of said bioadhesive polyphenolic protein areL-3,4-dihydroxyphenylalanine; the pH of said acidic solution is 4 orless; and the concentration said bioadhesive polyphenolic protein is10-250 mg/ml.
 2. A composition according to claim 1, wherein the pH ofthe acidic solution is 3 or less.
 3. A composition according to claim 1,wherein the pH of the acidic solution is 2.5 or less.
 4. A compositionaccording to claim 1, wherein the acidic solution comprises an organicacid.
 5. A composition according to claim 1, wherein the acidic solutioncomprises an inorganic acid.
 6. A composition according to claim 1,wherein the acidic solution comprises an acid chosen from the groupconsisting of citric acid, acetic acid, and ascorbic acid.
 7. Acomposition according to claim 1, wherein the concentration of thebioadhesive polyphenolic protein is in the range of 10-150 mg/ml.
 8. Acomposition according to claim 1, wherein at least one of the surfacesto be attached or the surface to be coated is a biological surface. 9.Use of a composition according to claim 1, wherein at least one of thesurfaces to be attached or the surface to be coated is a non-biologicalsurface.
 10. A composition according to claim 1, wherein 6-30% of theamino acid residues of said bioadhesive polyphenolic protein areL-3,4-dihydroxyphenylalanine.